The long range goals of this project are to understand structure- function relationships of the ADP -ribosylating exotoxins at the level of 3-dimensional crystallographic structure. The work described is focused on diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A (ETA), two related toxins that block protein synthesis by ADP-ribosylation of Elongation Factor-2 (EF- 2). With respect to DT, studies will be performed: (i) to characterize the active site, by combining site-directed mutagenesis with various biochemical and biophysical methods; (ii) to characterize the interaction of fragment A with EF-2 by new approaches, including the use of diphthamide synthesized by organic chemistry, and new assays for detecting interaction of EF-2 with active toxin fragments; (iii) to select toxin mutants that are defective in membrane-insertion and/or channel- formation at acidic pH, by means of a new positive selection procedure in E. coli; (iv) to explore a new method to study translocation of the enzymically active fragment A moiety of DT to the trans face of planar lipid bilayers; and (v) to determine the 3-dimensional structures of monomeric native DT, an enzymically active fragment (fragment A), and mutant forms of particular interest. With respect to ETA, studies are proposed which largely parallel those describe above for DT. Results will be interpreted in terms of a 3.0 A crystallographic structure of ETA determined recently, and an emerging 3-dimensional structure of DT-dimer at similar resolution. These studies are pertinent to a variety of problems in biomedical science, including mechanisms by which proteins penetrate membranes, routes to new vaccines, and mechanisms of directing toxic proteins to specific cells.